New technologies have been developed to determine base sequences of DNA's and RNA's.
In a method utilizing electrophoresis, which is usually used at present, a cDNA fragment sample, which is synthesized in advance from a DNA fragment or an RNA sample for sequence determination by performing a reverse transcription reaction, is prepared, a dideoxy reaction is performed by the well-known Sanger method, and then electrophoresis is performed to measure and analyze a pattern of separated base ladders.
On the other hand, in recent years, a method has been proposed for immobilizing many DNA fragments as samples on a substrate to determine sequence information of many fragments in parallel.
In Non-Patent Literature 1, microparticles are used as carriers for supporting DNA fragments to perform PCR's on the microparticles. After that, the microparticles supporting PCR-amplified DNA fragments are put into a plate provided with many holes, a diameter of which is matched to a size of the microparticles, to read out by a pyrosequence method.
Also, in Non-Patent Literature 2, using the microparticles as supports for supporting DNA fragments, PCR's are performed on the microparticles. After that, the microparticles are scattered and immobilized on a glass substrate, enzyme reactions (ligations) are performed on the glass substrate to let substrates with fluorescent dyes to be incorporated, and sequence information of each fragment is obtained by performing fluorescence detection.
Further, in Non-Patent Literature 3, many DNA probes having the same sequence have been immobilized on a substrate. Also, after scission of a DNA sample, an adapter sequence of a strand complementary to the DNA probe sequence is added to the terminal of each DNA sample fragment. By subjecting these to hybridization on the substrate, the sample DNA fragments are immobilized one molecule by one molecule randomly on the substrate. In this case, after performing DNA elongation on the substrate to let substrates with fluorescent dyes to be incorporated, washing off of unreacted substrates and fluorescence detection are preformed so that sequence information of sample DNA's is acquired.
As described above, a method for immobilizing many DNA fragment samples on a substrate and determining sequence information of many fragments in parallel has been developed and been put into practical use.